Making Octahedral Particles
Polyomavirus capsids are polymorphic under different chemical conditions. The protein of interest, VP1, is the primary building block of the capsid. Five VP1 proteins come together and form highly stable structures called pentamers. The number of pentamers that associate adjacently determines the curvature and symmetry of the viral capsid. Only one form is found in infectious virions, and the structure of this native form has been well studied. However, no high-resolution structure of the octahedral capsid currently exists. The goal of my research is to solve the structure of the octahedral capsid by electron microscopy and image classification. Since many particles are necessary for electron microscopy to be successful, I have produced and isolated these octahedral particles to provide necessary data. This octahedral capsid is created through a procedure beginning with purification of the VP1 protein. A recombinant plasmid containing DNA for a protein tag linked to VP1 is inserted into E coli which then produces this protein with autoinduction. The cells are lysed and VP1 is purified by selection for the protein tag in a flow column, where VP1 is then cleaved from the tag. This purified protein is then exposed to three distinct buffers that give the conditions necessary to make the octahedral particles.